Cloning, distribution, and effects of growth regulation of MC3R and MC4R in red crucian carp (Carassius auratus red var.)

Background Melanocortin-3 and -4 receptors (MC3R and MC4R), G protein-coupled receptors, play vital roles in the regulation of energy homeostasis. To understand the functions of mc3r and mc4r in the energy homeostasis of red crucian carp (Carassius auratus red var., RCC), we cloned mc3r and mc4r, analyzed the tissue expression and localization of the genes, and investigated the effects of knockout of mc3r (mc3r +/-) and mc4r (mc4r +/-) in RCC. Results The full-length cDNAs of RCC mc3r and mc4r were 1459 base pairs (bp) and 1894 bp, respectively. qRT-PCR indicated that mc3r and mc4r were profusely expressed in the brain, but lower expressed in the periphery tissues. ISH revealed that mc3r and mc4r were located in NPP, NPO, NAPv, NSC, NAT, NRL, NLTl, and NLTp of the brain, suggesting that mc3r and mc4r might regulate many physiological and behavioral aspects in RCC. To further verify the roles of mc3r and mc4r in energy homeostasis, the mc3r+/- and mc4r+/- fish were obtained by the CRISPR/Cas9 system. The average body weights, total lengths, body depths, and food intake of mc4r+/- fish were significantly higher than those of mc3r+/- and the normal wild-type (WT) fish, but there was no difference between the mc3r+/- and WT fish, indicating that the RCC phenotype and food intake were mainly influenced by mc4r but not mc3r. Interestingly, mc4r+/- fish displayed more visceral fat mass than mc3r+/- and WT fish, and mc3r+/- fish also exhibited slightly more visceral fat mass compared to WT. RNA-seq of the liver and muscle revealed that a large number of differentially expressed genes (DEGs) differed in WT vs. mc3r+/- , WT vs. mc4r+/- , and mc3r+/- vs. mc4r+/- , mainly related to lipid, glucose, and energy metabolism. The KEGG enrichment analysis revealed that DEGs were mainly enriched in pathways such as steroid biosynthesis, fatty acid metabolism, fatty acid biosynthesis, glycolysis/gluconeogenesis, wnt signaling pathway, PPAR signaling pathway, and MAPK signaling pathway, thereby affecting lipid accumulation and growth. Conclusion In conclusion, these results will assist in the further investigation of the molecular mechanisms in which MC3R and MC4R were involved in the regulation of energy homeostasis in fish.

MC3R and MC4R are mainly expressed in the central nervous system (CNS) and play critical roles in regulating energy homeostasis.To our knowledge, mutations of mc3r or mc4r in mice result in obesity (3,4).The mc4r knockout mice show decreased energy expenditure, increased food consumption, increased fat mass, increased somatic growth, and substantial insulin resistance (3,5).It is of note that the obesity phenotype of mc3r knockout mice are different from mc4r knockout mice, mc3r knockout mice do not exhibit significantly increased food intake, increased weight, and substantial insulin resistance, while they have increased fat mass and feed efficiency (4,6).Recent data suggest that mc3r knockout mice are involved in triglyceride synthesis and hepatic lipogenesis (7).Likely, mutations of mc3r or mc4r in human are associated with obesity (5, 8,9).
The relative expression of mc3r and mc4r has also been found in several peripheral tissues in addition to the central nervous system, indicating other potential physiological functions in the periphery with reproduction, cardiovascular, and immune response (1,(9)(10)(11)(12)(13).The MC3R and MC4R are primarily coupled to the stimulatory G protein (Gs) to stimulate the adenylyl cyclase activity, which will increase intracellular cAMP production to trigger downstream signaling (11,14).
Red crucian carp (Carassius auratus red var.) is an ornamental fish with vivid red color and accounts for important proportion of freshwater aquaculture production worldwide.In this study, we investigated the physiological functions of MC3R and MC4R in red crucian carp by gene cloning, tissue distribution, and localization.To further verify the roles of MC3R and MC4R in regulating energy homeostasis and growth, we obtained the mc3r and mc4r knockout RCC and compared the difference of growth performance, lipid accumulation, DEGs, and KEGG pathways among different types.These findings laid the foundation for future physiological studies of teleosts MC3R and MC4R.
2 Materials and methods

Animal and ethics statement
All animal experiments were approved by the Animal Care Committee of Hunan Normal University and the Administration of Affairs Concerning Experimental Animals of China.Red crucian carp was collected from the Engineering Research Center of Polyploid Fish Reproduction and Breeding of the Ministry of Education at Hunan Normal University.

Gene cloning and sequence alignment
Red crucian carp was anesthetized before decapitation and the tissues were excised and stored at −80°C.Total RNA was purified by Trizol ™ Reagent (Invitrogen, USA).The first-strand cDNA was synthesized by PrimeScript RT reagent Kit (TaKaRa, Japan).Primers, designed using Primer Premier 5.0 (Supplementary Table 1), were used to obtain full-length cDNA via PCR and touch-down PCR with TaKaRa LA Taq ® .The PCR products were separated through 1.2% agarose gels, purified by SanPrep Column DNA Gel (Sangon, China), subcloned into the PMD18-T, and then sequenced (Sangon).

Quantitative reverse transcription PCR
To explore the tissue distribution of these genes, the medulla, mesencephalon, cerebellum, olfactory bulb, telencephalon, hypothalamus, pituitary, spleen, muscle, gonads (ovary and testis), gill, heart, liver, kidney, head kidney, and skin were obtained from three males (45.27 ± 4.79 g) and three females (46.33 ± 3.17 g), respectively.Primers were designed by AlleleID 6 and selected in qRT-PCR with 95%-105% amplification efficiency and single melting curve, and bactin was used as the internal control (Supplementary Table 1).The qRT-PCR was carried out by a Prism 7,500 Sequence Detection System (ABI, USA).The 10-mL mixture of the reaction consisted of 5 mL of SYBR green PCR Master Mix (TaKaRa), 0.5 mL of each primer, 3 mL of ddH 2 O, and 1 mL of diluted cDNA sample.The qRT-PCR procedure was set as follows: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 61°C for 45 s.The experimental samples were added to a 96-well plate repeated thrice.The relative expression of genes was calculated using the 2 −DDCt method (22).

In situ hybridization
Primers (Supplementary Table 1) for the in situ hybridization (ISH) were designed by Primer Premier 5.0.Antisense digoxigenin (DIG)-labeled probes were synthesized through in vitro transcription using MAXIscript ™ T7 In Vitro Transcription Kit (Invitrogen, USA) and purified by LiCl precipitation as described in the title (23).The probes were stored at −80°C.
The RCC brain was fixed in 4% paraformaldehyde (PFA, Sangon) for a night at 4°C.Next, the tissue was dehydrated using a series of graded sucrose solutions (15%, 20%, and 30%) and embedded in Tissue-Tek O.C.T. Compound ® (Sakura, USA).Then, the embedded tissues were cut using a freezing microtome (Leica CM3050 S Cryostat, Germany) into 20-mm-thick sections according to the instruction manual.
The ISH experiment needs to maintain an RNase-free environment.Prior to hybridization, the sections were washed with 1×PBST at room temperature (RT) for 5 min, treated with 5 mg/mL proteinase K at RT for 5 min, fixed with 4% PFA at RT for 10 min, and prehybridized (prehybridization solution was mixed as follows: 50 mL of deionized formamide, 25 mL of 20 × SSC, 920 mL of 1 M citric acid, 1 mL of 0.5 M EDTA, 500 mL of 20% Tween-20, 100 mL of 50 mg/mL heparin, 100 mL of 50 mg/mL tRNA, and 22.38 mL of DEPC-H2O) at 60°C for 4 h.Then, the sections were hybridized (hybridization solution was prepared by adding the RNA probe into prehybridization solution) at 60°C for 16 h.Post-hybridization, the sections were washed with 50% deionized formamide at 50°C for 15 min, 2×SSCT at RT for 15 min, and incubated with 2 mg/mL RNase A (Thermo Fisher Scientific, USA) buffer at RT for 30 min.Then, they were washed with 2×SSCT, 0.2×SSCT, and 1×PBST for 10 min, respectively.Next, the sections were blocked with blocking buffer at RT for 2 h and then incubated with AP-conjugated anti-DIG antibody (Roche, Germany, 1:3,000 diluted in the blocking buffer) overnight at 4°C.The sections were stained with NBT/BCIP stock solution (Roche, Germany) and the hybridization signals were detected using light microscopy (Olympus, Japan).The negative control was incubated with PBS instead of probes.

Gene editing via the CRISPR/ Cas9 system
The CRISPR/Cas9 system was made up of Cas9 protein and guide RNA (gRNA) (24).gRNAs were designed and generated using the Maxiscript T7 PCR-based method.The gRNAs targeting mc3r and mc4r were designed using the online software tool (http:// zifit.partners.org/ZiFiT)and two gRNAs of each gene were selected to ensure the knockout efficiency.Taking the DR274 plasmid as a template, the gRNAs were synthesized with corresponding primers (Supplementary Table 1) using Takara EX Taq (TaKaRa, Japan).The PCR products were confirmed with 1.2% agarose gel and used as templates in subsequent in vitro transcription reactions following the manufacturer's guidelines of TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific, USA) for synthesizing the gRNAs.
The TrueCut ™ Cas9 Protein v2 was obtained from Thermo Fisher Scientific.The injection solutions were prepared by mixing Cas9 Protein and gRNAs, and then the mixture was incubated for 5 min on ice before being injected into the two-cell stage via a microinjector (PV830, WPI, USA).The final concentrations of gRNAs and Cas9 Protein were 200 ng/mL and 300 ng/mL, respectively.
Fin-clip samples of each fish were collected in sterile Eppendorf tubes and extracted genomic DNA using Tissue DNA Kit (Omega, USA) for mutation examination.The primers (Supplementary Table 1) for PCR amplification were encapsulated all possible mutation sites.Then, the amplified products were separated, purified, and sequenced as above.

Feeding trial
For this experiment, 6-month-old WT (n = 18, 10.28 ± 2.18 g), mc3r +/− (n = 18, 10.38 ± 2.27 g), and mc4r +/− (n = 18, 16.80 ± 4.68 g) fish were individually placed into nine tanks (six fish each) with the same feeding environment, and each type of fish was divided into three replicates.The fish were fed once a day.The feeding trial was carried out for 12 days and food intake was recorded daily.Food intake was calculated as the difference between the initial dry weight and residual dry weight.

Transcriptome sequencing
Total RNA was extracted from liver and muscle samples of treatment and control groups for transcriptome sequencing.The cDNA libraries were constructed using the NEBNext ® UltraTM RNA Library Prep Kit for Illumina ® (NEB, USA) according to manufacturer instructions.Then, the cDNA libraries were sequenced with Illumina HiSeq ™ sequencing platform.The fastp v 0.19.3 was used to remove the low-quality bases and empty reads.Next, HISAT v2.1.0was used to construct the index and compare clean reads to the reference genome (https://www.ncbi.nlm.nih.gov/nuccore/1764596402).The differential expressions of two groups were analyzed via DESeq2 v1.22.1.Genes with |log2Fold Change| ≥ 1 and false discovery rate (FDR) < 0.05 were defined as differentially expressed genes (DEGs).The DEGs enrichment analysis was performed based on the hypergeometric test.The Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) were performed based on the pathway and GO term, respectively.

Statistical analysis
DNAMAN version 5.0 was used to analyze multiple sequence alignment.Phylogenetic tree was constructed with MEGA 5 software.SPSS 19.0 software was used to calculate all data that were presented as the mean ± SEM.One-way ANOVA was used to analyze statistically significant differences in gene expression between multiple groups.The significance of differences in the average body weights, total lengths, body depths, food intake, visceral fat mass, and gene expression of mutated and control fish were determined by Student's t-test.p < 0.05 was considered as statistically significant.

Tissue expression of RCC mc3r and mc4r
The qRT-PCR was used to analyze the relative expression of RCC mc3r and mc4r.The results reported that mc3r and mc4r were mainly expressed in the brain, and moderately expressed in peripheral tissues (Figure 3).In female RCC, mc3r was profusely expressed in the hypothalamus and pituitary gland but expressed at low levels in other tissues (Figure 3A).In male RCC, the highest expression of mc3r was observed in the hypothalamus, and also partially expressed in the olfactory bulb, telencephalon, spleen, testis, and head kidney (Figure 3B).
Similar to RCC mc3r expression.In female RCC, mc4r was highly expressed in the olfactory bulb, hypothalamus, telencephalon, mesencephalon, and ovary (Figure 3C).In male RCC, the relative expression of mc4r was higher in the hypothalamus, olfactory bulb, telencephalon, and followed by testis (Figure 3D).ISH was used to further determine the localization of mc3r and mc4r in the RCC brain with antisense probes, with the PBS used as the negative control.
Both of the cell groups expressing mc3r and mc4r mRNAs were detected in several parts of the preoptic area and tuberal hypothalamus.In the preoptic area, mc3r-expressing and mc4r-expressing neurons were localized in the periventricular part of the preoptic nucleus (NPP), magnocellular neurons of the preoptic nucleus (NPO), the anterior periventricular nucleus (NAPv), and the suprachiasmatic nucleus (NSC), in which the expressions adopt a periventricular disposition (Figures 4A, B).Within the tuberal hypothalamus, positive mc3rlabeled and mc4r-labeled cells were evident in the anterior tuberal nucleus (NAT), the lateral recess nucleus (NRL), the posterior part of the lateral tuberal nucleus (NLTp), and the lateral part of the lateral tuberal nucleus (NLTl) (Figures 4D, E).No signals were found in the negative control (Figures 4C, F).

Generation of mc3r and mc4r mutations by the CRISPR/Cas9 system
To verify the physiological functions of mc3r and mc4r in RCC energy homeostasis, the CRISPR/Cas9 system was used to mutate the RCC mc3r and mc4r.As we all know, genomic mutations contain either or all of the deletion, insertion, and substitution in the target areas at different levels.The PCR and Sanger sequencing revealed that multi-peaks were found in mc3r-gRNA 2 and mc4r-gRNA 1 , the targeted areas (Figures 5, 6).The gene cloning results of mutant mc3r and mc4r both had 2 different levels of mutations (Figures 5D, 6D).There were two types of mutations in the mutant mc3r-gRNA 2 group, including a type with 40-bp deletion, and another type with 3-bp deletion but 7-bp insertion (Figure 5D).Mutated fishes from the mc4r-gRNA 1 group also exhibited two types of mutations, consisting of a type of 179-bp deletions and a type of 4-bp insertion (Figure 6D).The mutation rates were 32.3% in the mc3r mutation and 35.5% in the mc4r mutation.The survival rates of WT, mc3r +/− , and mc4r +/− were 82.1%, 80.5%, and 57.7%, respectively.

Transcriptome analysis and qRT-PCR verification
Transcriptome analysis of RCC WT, mc3r mutation, and mc4r mutation were performed to explain the mechanism of mc3r and mc4r in regulating energy homeostasis.The raw reads of liver and muscle were submitted to NCBI under accession PRJNA1018679 and PRJNA1018689, respectively.

Go enrichment analysis of DEGs
GO annotation of DEGs was obtained by Blast2GO, and classified into three categories: biological process (BP), cellular component (CC), and molecular function (MF) (Supplementary Figure 2).Moreover, GO enrichment analysis of the WT vs. mc3r +/− , WT vs. mc4r +/− , and mc3r +/− vs. mc4r +/− in liver and muscle was performed, respectively, and the top 20 enrichment terms are shown in Figure 10.
In the liver, GO annotation of the three groups showed that most DEGs of the three groups were all annotated into BP and MF (Supplementary Figures 2A-C).GO enrichment analysis of WT vs. mc3r +/− showed that the enriched terms included organic acid transmembrane transporter activity, carboxylic acid transmembrane transporter activity, L-amino acid transmembrane transporter activity, arachidonate-CoA ligase activity, and long-chain fatty acid-CoA ligase activity (Figure 10A).In the WT vs. mc4r +/− , the sterol biosynthetic process, secondary alcohol biosynthetic process, cholesterol biosynthetic process, steroid biosynthetic process, and sterol metabolic process were the most enriched (Figure 10B).In mc3r +/− vs. mc4r +/− , secondary active sulfate transmembrane transporter activity, transferase activity, transferring nitrogenous groups, sulfate transmembrane transporter activity, iron ion binding, and organic acid transmembrane transporter activity were the most enriched (Figure 10C).
In the muscle, GO annotation of the three groups showed that most DEGs of the three groups were all annotated into BP (Supplementary Figures 2D-F).GO enrichment analysis of the WT vs. mc3r +/− showed that the enriched terms included unfolded protein binding, protein folding, protein refolding, protein folding chaperone, and myosin filament (Figure 10D).In the WT vs. mc4r +/− , the enriched terms included proteasome complex, proteasome accessory complex, endopeptidase complex, proteasome regulatory particle, and peptidase complex (Figure 10E).In mc3r +/− vs. mc4r +/− , striated muscle thin filament, myofilament, troponin complex, myosin filament, and protein refolding were the most enriched (Figure 10F).

DEGs verification by qRT-PCR
To verify the dependability of transcriptome data, the DEGs associated to lipid metabolism, glucose metabolism, and amino acid metabolism were selected for qRT-PCR validation in the liver and muscle of the three groups.In the liver, LDHA, eno1, eno3, tgfbr2, nampta, p4ha1, fbp1b, FACL4, and aldob were used for qRT-PCR validation (Figure 12).In muscle, DUSP8, irak4, acadl, rps24, mgll, psma4, psma6, psmb4, and cpt2 were used for qRT-PCR validation (Figure 13).The primers were designed based on transcriptome sequencing data (Supplementary Table 1).Our results showed that all the relative expression levels of the above genes had a similar trend with transcriptome data, indicating the dependability of transcriptome sequencing.

Discussion
In the present study, we cloned the full-length cDNAs of mc3r and mc4r in red crucian carp, which were 1,459 bp and 1,894 bp, respectively (Figures 1A and 2A).The mc3r had a 984-bp ORF encoding a putative protein of 327 amino acids and the mc4r had a 981-bp ORF encoding a putative protein of 326 amino acids (Figures 1A and 2A).Multiple sequence alignment revealed that RCC MC3R and MC4R both had seven TMDs and several conserved motifs as other species (Figures 1A and 2A, and Supplementary Figure 1).Phylogenetic analysis showed that MC3R and MC4R clustered with teleost MC3Rs and MC4Rs, individually (Figures 1B and 2B), consistent with previous studies (8,9,11,25).These data indicated that RCC MC3R and MC4R shared high identity levels with MC3Rs and MC4Rs of other species, respectively.
We observed that RCC mc3r and mc4r were highly expressed in the brain, especially in the hypothalamus (Figure 3), indicating their roles in regulating energy homeostasis (9,11,25,26).In addition, mc3r and mc4r were widely expressed in peripherical tissues, especially in the ovary and testis, indicating that they might play a vital function in regulating reproduction.Of note, several studies have confirmed the point that MC4R is associated with reproduction in various species (7,13,(27)(28)(29)(30).However, no data prove that MC3R is involved in regulating reproduction.
To further verify the functions of the central MC3R and MC4R in RCC, we detected the neuronal localization of mc3r and mc4r in the brain by ISH.In the brain, we found that the two receptor transcripts were observed in almost areas of the brain, such as the preoptic area, ventral thalamus, tuberal hypothalamus, and hypothalamic inferior lobe, and the positive signal of mc4r was stronger than mc3r (Figure 4).To our knowledge, the CNS is the center of appetite regulation, involved in regulating feeding, nutrient partitioning, and energy expenditure as in mammals (21,31,32).Previous reports have indicated that hormones involved in food intake such as NPY, AGRP, and were highly expressed in brain (33)(34)(35).Briefly, the mc3r and mc4r highly expressed in the brain might be associated with appetite regulation like the above hormones.We found that mc4r was extensively expressed in NPP, NPO, NAPv, NAT, NRL, NLTl, and NLTP (Figures 4B, E), consistent with the previous studies in rats and teleost (36-39).Evidence also shows that mc4r is expressed on NPY, GHRH, and AgRP neurons (40), and NPY could stimulate GH secretion (41).Similarly, mc3r was also detected in the same areas of the brain as the distribution of mc4r (Figures 4A, D).The distribution of mc3r is also observed in the hypothalamic arcuate nucleus of mice (42) and mc3r is expressed on POMC, GHSR, and NPY neurons, which are associated with feeding and growth (42,43).In cyprinid fish, the NPP and NPO are primary corticotrophin-releasing hormoneproducing areas (36,44).The NLT of fish is regarded as a homologue of mammalian arcuate nucleus, which is a crucial receptor of feeding in mammals (40,45).MC3R and MC4R co- expressed in these areas of the brain indicated that MC3R and MC4R might play crucial roles in regulating feeding and growth of RCC.
To further investigate the effects of MC3R and MC4R in growth, food intake, and energy homeostasis, the RCC mc3r +/− and mc4r +/− were generated by the CRISPR-Cas9 system with microinjection of gRNAs targeting the RCC mc3r and mc4r in conjunction with Cas9 protein.We found that the average body weights, total lengths, body depths, and food intake of mc4r +/− fish were significantly higher than WT and mc3r +/− fish, but there was no significant difference between WT and mc3r +/− fish at 6 mah (Figures 7A-D), indicating that RCC MC4R might play more important roles in growth and food intake than MC3R, consistent with previous studies (3-6, 19, 36).In addition, it is more interesting that RCC mc4r +/− displayed more visceral fat mass than mc3r +/− and WT.RCC mc3r +/− also exhibited slightly more visceral fat mass compared to WT (Figures 7E-H), indicating that MC3R and MC4R played an essential role in lipid accumulation and the role of MC4R was more significant than that of MC3R, consistent with previous studies in mice (3,46).The mc4r mutants have been researched in several species including fish, pigs, and chickens (3,(47)(48)(49)(50)(51)(52).In channel catfish, the body weights of mc4r-deficient fish exhibited 20%-38% improvement compared to WT (49).Holland's carp containing SNP in the mc4r gene have more predominant growth traits than WT (52).Pigs and chickens containing SNPs in the mc4r gene also have more predominant growth traits than WT (48,51).In addition, previous studies have proved that the body weights and fats of mc3r-deficient pigs were higher than WT (53).Chickens and blue foxes containing SNPs in the mc3r gene have more predominant growth traits than WT (54, 55).It can be seen that our data were consistent with those of previous studies.These data suggested that MC3R and MC4R were involved in growth regulation and food intake, but MC4R may play more important roles than MC3R.The improved growth in mc3r-and mc4rdeficient RCC might provide a new strategy for aquaculture.
The liver plays crucial roles in regulating glycogen storage, protein synthesis, and hormone production, and muscle is a major effector organ in fish (56, 57).Thus, we obtained and further analyzed the transcriptome sequences of liver and muscle in WT and mc3rand mc4r-deficient RCC.Results show that a large of DEGs were found in the liver and muscle of WT vs. mc3r +/− , WT vs. mc4r +/− , and mc3r +/− vs. mc4r +/− , and the DEGs mainly enriched in these pathways were related to growth, development, and energy metabolism (Figures 8, 9, and 11).In the liver, we found that mc3r deficiency could lead to the differential expression of the genes compared to WT like FACL4, fabp7a, LDHA, mthfr, and odc1 in RCC.The FACL4 plays an important role in lipid biosynthesis and fatty acid degradation (58).The fabp plays an essential role in intracellular lipid trafficking, and the upregulation of fabp7a can cause lipid accumulation (59)(60)(61).The LDHA-deficient mice exhibit moderately elevated body fat (62).The mthfr polymorphisms correlate with high homocysteine levels and subsequent insulin resistance and will cause overweight (63,64).The odc1-deficient fruit fly exhibits increased number of fat cells and triglycerides (65).mc4r deficiency also led to the differential expression of the genes compared to WT like sc5d, dhcr7, gamt, eno1, eno3, and acat2 in RCC.The sc5d and gamt play crucial roles in growth, lipid synthesis, and cellular metabolism (66,67).The dhcr7 is involved in steroid synthesis (68).The acat2 is a key gene for fatty acid anabolism (69).The eno1 has been found to be involved in the development, growth, and reproduction of organisms (70), and the eno3 is well known for its functions in energy and lipid metabolism (71).In the mc3r +/− vs. mc4r +/− , there were also some DEGs related to growth, lipid, and glucose metabolism like pck1, foxo1a, gatm, acot12, and cdo1 in RCC.The pck1 is a key gene for lipid metabolism and glucose homeostasis (72).The foxo1a is thought to be involved in controlling food intake (73).The gatm plays vital roles in lipid metabolism and energy homeostasis (67).The acot12 and cdo1 are suggested to be involved in regulating lipogenesis (74,75).
In muscle, we found that mc3r deficiency could lead to the differential expression of the genes compared to WT like DUSP8, irak4, gpam, mgll, and lipg in RCC.The DUSP8-deficient mice can increase the energy expenditure and thus affect the body weight (76).The irak4 can regulate the secretion of glucagon (77).The gpam is an enzyme in lipid metabolism and is thought to be influenced by the synthesis of triglyceride, cholesterol, and fatty acid contents in bovine mammary epithelial cells (78).The mgll is suggested to be involved in the regulation of lipid and biosynthesis and glycerolipid metabolism (79).The lipg is a member of the triglyceride lipase family and is integrally involved in lipid absorption, transport, and metabolism (80).mc4r deficiency also led to the differential expression of the genes compared to WT like psma4, psma6, psmb4, acadl, cpt1, cpt2, and mat2b in RCC.The psma4 can regulate insulin sensitivity (81).The psma6, mat2b, and psmb4 play essential roles in adipogenesis (82, 83).The acadl, cpt1, and cpt2 are thought to be involved in lipolysis (84)(85)(86).In mc3r +/− vs. mc4r +/− , there were also some DEGs related to growth, lipid, and glucose metabolism like nampta, SGK1, irs1, and IGF-1R in RCC.nampta is involved in regulating feeding and energy homeostasis in goldfish (87).SGK1 may be associated with insulin secretion and obesity (88).The decreased expression of irs1 may cause insulin resistance (89).The IGF-1R mutation has been suggested to affect skeletal development and growth in sheep (90).These data suggested that mc3r and mc4r mutations affected the differential expression of these genes involved in growth, lipid, and glucose metabolism, consistent with the above results regarding growth performance and lipid accumulation after mc3r and mc4r mutations in RCC.
In summary, we cloned the full-length cDNAs and analyzed the relative expression of mc3r and mc4r in RCC.Both genes were primarily present in the CNS, and widely present in the periphery.We also detected the localizations of mc3r and mc4r in the brain, and we found that the mc3r-expressing and mc4r-expressing neurons were localized in NPP, NPO, NAPv, NSC, NAT, NRL, NLTP, and NLTl, which were involved in regulating feeding and growth.The average body weights, total lengths, body depths, and food intake of mc4r +/− fish were significantly higher than those of mc3r +/− and WT fish, but there was no significant difference between mc3r +/− and WT fish.However, it is more interesting that RCC mc4r +/− displayed more visceral fat mass than mc3r +/− and WT.RCC mc3r +/− also exhibited slightly more visceral fat mass compared to WT.The data of RNA-seq showed that both mc3r and mc4r knockout affected the differential expression of many genes involved in growth, lipid, and glucose metabolism, indicating that mc3r and mc4r play vital roles in growth, lipid accumulation, lipidolysis, and insulin resistance.These findings laid the foundation for future physiological studies on the functions of mc3r and mc4r that might provide new strategies for improving the growth and aquaculture of teleost.

FIGURE 9
FIGURE 10GO enrichment analysis of the DEGs in liver and muscle (n = 3).(A) GO enrichment analysis of the DEGs between WT and mc3r +/− in liver.(B) GO enrichment analysis of the DEGs between WT and mc4r +/− in liver.(C) GO enrichment analysis of the DEGs between mc3r +/− and mc4r +/− in liver.(D) GO enrichment analysis of the DEGs between WT and mc3r +/− in muscle.(E) GO enrichment analysis of the DEGs between WT and mc4r +/− in muscle.(F) GO enrichment analysis of the DEGs between mc3r +/− and mc4r +/− in muscle.The vertical coordinate represents the GO term.The horizontal coordinate represents the Rich factor.The larger the Rich factor, the greater the degree of enrichment.The larger the dot, the greater the number of DEGs enriched in the GO term.The redder the color of the dot, the more significant the enrichment.

11 KEGG 12
FIGURE 11 KEGG enrichment analysis of DEGs in liver and muscle (n = 3).(A) KEGG enrichment analysis of the DEGs between WT and mc3r +/− in liver.(B) KEGG enrichment analysis of the DEGs between WT and mc4r +/− in liver.(C) KEGG enrichment analysis of the DEGs between mc3r +/− and mc4r +/− in liver.(D) KEGG enrichment analysis of the DEGs between WT and mc3r +/− in muscle.(E) KEGG enrichment analysis of the DEGs between WT and mc4r +/− in muscle.(F) KEGG enrichment analysis of the DEGs between mc3r +/− and mc4r +/− in muscle.The vertical coordinate represents the GO term.The horizontal coordinate represents the Rich factor.The larger the Rich factor, the greater the degree of enrichment.The larger the dot, the greater the number of DEGs enriched in the GO term.The redder the color of the dot, the more significant the enrichment.
bValues with different superscripts in the same column are significantly different (p < 0.05).Data are shown as the mean ± SEM (n = 8).mah, months after hatching.